DESCRIPTION: (Applicant's Abstract) Epithelial stem cells are responsible for the homeostasis of self-renewing epithelia. Moreover they play a central role in wound repair and are the target cell for tumor initiation. My work during the past decade has contributed to the identification of stem cells within the corneal epithelium. Isolation of limbal epithelial stem cells will greatly facilitate their characterization. Furthermore, isolation of pure populations of limbal stem cells will provide the ultimate starting material for epithelial cells needed for transplantation in corneal reconstruction. The lack of specific markers for epithelial stem cells has become a major impediment in their isolation and subsequent biochemical characterization. The goal of this project is to identify specific markers for limbal epithelial stem cells. Towards this end we will: 1) use single cell technology in combination with molecular biology to obtain mRNA from limbal epithelial stem cells and corneal epithelial transit amplifying (TA) cells; 2) use suppressive subtractive hybridization (SSH) and cDNA microarrays to identify genes that are differentially expressed in limbal epithelial stem cells versus corneal epithelial TA cells; and 3) characterize the limbal epithelial stem cell-specific genes and investigate their potential function(s), focusing on those genes that are associated with the cell surface. Data obtained from these studies should lead to the isolation of pure populations of limbal epithelial stem cells. This will facilitate the biochemical characterization of these cells, and will provide a better understanding of the mechanisms involved in the self-renewal of stem cells, and in the mechanisms of limbal and corneal epithelial growth and differentiation.